A protosilencer of subtelomeric gene expression in Candida glabrata with unique properties

Abstract

Adherence to host cells is an important step in the pathogenicity of the opportunistic fungal pathogen Candida glabrata. This adherence is mediated by some members of the large family of cell wall proteins encoded by the EPA (Epithelial Adhesin) genes present in the C. glabrata genome. The majority of the EPA genes are localized close to different telomeres in C. glabrata, resulting in a negative regulation of transcription of these genes through chromatin-based subtelomeric silencing. In vitro, adherence to epithelial cells is mainly mediated by Epa1, the only member of the EPA family that is expressed in vitro. EPA1 forms a cluster with EPA2 and EPA3 at the subtelomeric region of telomere E(-R). EPA2 and EPA3 are subject to silencing that propagates from this telomere in a process that depends on the Sir2, -3, -4, and Rif1 proteins, but surprisingly not on the yKu70 and yKu80 proteins. Here we describe that the yKu70/yKu80-independent silencing of telomere E(-R) is due to the presence of a cis-acting protosilencer (Sil2126) located between EPA3 and the telomere. This element can silence a reporter gene when placed 31.9 kb away from this telomere, but not when it is removed from the telomere context, or when it is placed near other telomeres, or inverted with respect to the reporter. Importantly, we show that the cis-acting Sil2126 element is required for the yKu70/80-independent silencing of this telomere, underscoring the importance of cis-elements for repressive chromatin formation and spreading on some telomeres in C. glabrata.

Figure 1

The cis-acting silencing element Sil2126 is composed of at least two functional modules and requires Sir2, Sir3, Rap1, Rif1, but not the yKu70/80 heterodimer. (A, top) Schematic representation of the E_-R telomere showing the Sil2126 element (represented by a shaded rectangle labeled 2126), and the nucleotide position of its 5′ end (4.52 kb from the telomere). Arrowheads indicate 5′ to 3′ direction of Sil2126. Lightly shaded arrows represent the different subtelomeric EPA genes and indicate their direction of transcription. (Middle) Map of the Sil-reporter system used to integrate at the SpeI site between ISC1 and HYR1, consisting of a PCR fragment containing the integration region, cloned immediately adjacent to the 5′ end of the Sil2126 element followed by the URA3 reporter gene. The SpeI recognition site (at −31.9 kb) used to linearize and integrate the vector is indicated. (Bottom) Schematic representation of the Sil2126-reporter system integrated between ISC1 and HYR1 (indicated by a thick solid line). The telomere is represented by solid rectangles labeled “Tel.” (B) Assessment of the level of silencing of serial 5′ and 3′ deletions of the Sil2126 element and precise deletions of the two modules identified. On the left side is shown the different constructs represented by open rectangles, used to assay the level of silencing of the URA3 gene conferred by the truncated versions of the Sil2126 element. Numbers on either side of the rectangles indicate the nucleotide position of the 5′ and 3′ ends of the truncated versions. The complete, original Sil2126 element is indicated by the shaded rectangle. On the right side is shown the level of silencing conferred by the truncated Sil elements tested as measured by growth on SC −ura and 5-FOA plates. Strains of C. glabrata containing the different truncated versions of the Sil element integrated between ISC1 and HYR1 were grown to stationary phase in YPD, and 10-fold serial dilutions in sterile water were made. Equal numbers of cells of each dilution were spotted onto SC −ura and SC plates containing 5-FOA. Plates were incubated at 30° for 48 hr and photographed. (C) Schematic representation of different combinations of Sil2126 elements at telomere E_R. (Top) Map of the E_-R telomere showing the original Sil2126 element at its native position (−4.52 kb from the telomere repeats) plus the integrated Sil2126-reporter system integrated at −31.9 kb (SpeI site) generating a duplication of Sil2126 element, indicated by Sil +/+ on the right side. (Middle) Integration of the negative control used consisting of the URA3 reporter integrated at −31.9 kb without Sil2126. This construct conserves the original Sil2126 at −4.52 kb and is indicated by sil −/+. (Bottom) Map of the construct containing the Sil2126-reporter system integrated at −31.9 kb of telomere E_-R and deletion of the original Sil element at −4.52 kb, indicated by Sil +/−. (D) Level of silencing of the Sil-URA3 reporter constructs (indicated to the left) in different mutants of the silencing machinery. The experiments were made as indicated in B.

Figure 2

Sil2126 requires the telomere E_-R context and is orientation dependent. (A) Dependence of the Sil element on the proximity to the telomere. Schematic representation of the Sil2126-reporter system is shown on the left side (Sil+). The schematic map of the negative control (sil−) consisting only of the URA3 reporter with no Sil2126 is not shown. Both of these constructs are integrated at two positions in telomere E_-R: at −31.9 kb (the URA3 gene starts at position −35 kb), lines 1 and 2; and integration at −50 kb (lines 3 and 4). Integration of the Sil+ or sil− constructs at an internal position in chromosome F (527 kb from the telomere F_R, lines 5 and 6). Integration of the constructs at an internal position in chromosome M (224 kb from the telomere M_-L, lines 7 and 8) and integration of the Sil+ at an internal site in chromosome L at 678 kb from telomere L_-L (line 9). On the right side, the growth phenotype on SC −ura and on 5-FOA plates is shown for each construct to assess the level of silencing. Strains carrying the corresponding Sil-reporter constructs integrated at the indicated chromosomal locations were grown to stationary phase in YPD and diluted and spotted on SC −ura and 5-FOA plates as described in Figure 1B. (B) Sil2126 is functional only in the context of telomere E_-R. Insertion of Sil+ and sil− reporter constructs in both orientations at −34 kb from telomere E_-L (lines 10–12) or at various distances from three other telomeres: C_-L, I_-L, and K_-R abolishes silencing activity of the element (lines 13–18). (C) Silencing activity of Sil2126 is orientation dependent. The orientation of the Sil2126 was inverted with respect to the URA3 reporter and integrated at −31.9 kb from the telomere E_-R (lines 19 and 21). Orientation of the entire module Sil2126-URA3 was inverted, resulting in inversion of both elements of the system with respect to the original orientation but maintaining the same relative position to each other (line 22). All the telomeres were drawn to the right of the sequences. The URA3 reporter and Sil2126 were flipped accordingly to show the correct relative orientation and order between each element and the telomere. Arrowheads indicate the 5′ to 3′ direction of Sil2126.

Figure 3

Subtelomeric silencing in telomere E_-R is abolished by the presence of a strong promoter between the telomere and the URA3 reporter. (A, top) Schematic representation of the subtelomeric region of chromosome E_-R. Open, shaded arrows indicate the five native genes present in this region and their respective direction of transcription. Inverted solid triangles indicate URA3 insertions at the positions shown with respect to the telomere. The shaded rectangle indicates the Sil2126 element at its original position. Arrowhead indicates the 5′ to 3′ direction of the element. (Middle) The hph cassette is excised from the deletion/insertion allele leaving one copy of the 35‐bp FRT site. (Bottom) Sil deletion/insertion allele was integrated in each strain carrying the corresponding URA3 reporter insertions at different positions throughout telomere E‐R. The hph gene driven by the strong promoter from PGK1 is inserted replacing Sil2126 thus placing a strong promoter close to the telomere. (B) Plate growth assay of strains containing the indicated URA3 reporter insertions at increasing distances from the telomere in the wild-type Sil2126 genetic background (Sil+), the sil simple deletion (silΔ), and sil deletion/insertion allele (silΔ::P_PGK1::hph) as indicated. Strains containing each of the URA3 reporter insertions in each of the three backgrounds were grown to stationary phase in YPD and spotted onto the indicated plates as described in Figure 1B.

Figure 4

yKu70/80 independence of subtelomeric silencing on chromosome E_-R requires the Sil2126 element. (A) Silencing of four different URA3 reporters (solid triangles) in the E_-R telomere as measured by the ability to grow on 5-FOA plates. Strains containing the indicated reporter insertions throughout the E_-R telomere (shown at the top of each panel) were used as recipients to introduce the following deletion alleles: silΔ, hdf1Δ (yKu70), hdf2Δ (yKu80), or double deletions hdf1Δ silΔ or hdf2Δ silΔ. All of these strains were grown to stationary phase and spotted onto the indicated plates as described in Figure 1B. (B) Sil2126 is not sufficient to confer yKu independence to the yKu-dependent telomere I_-R. The Sil-reporter system was integrated at position −2.1 kb from telomere I_-R where subtelomeric silencing depends on both yKu proteins. These strains were used as recipients to introduce hdf1Δ (yKu70), hdf2Δ (yKu80) alleles. The level of silencing was assessed in the same way as described in Figure 1B. (C) EPA3 transcription is strongly derepressed both when the Sil2126 element is deleted as well as one of the HDF genes (hdf1Δ or hdf2Δ) or when the strong promoter from PGK1 is inserted between the telomere and EPA3. We used a sir3Δ strain as control since we have previously shown that EPA3 is derepressed in the absence of Sir3. All strains were grown to stationary phase and total RNA was isolated and used for RT–PCR (see Materials and Methods). Lane 1 shows genomic DNA used as positive control for PCR. Actin-specific primers were used as internal control for RT–PCR. A negative control without RT was also made and no bands were obtained (not shown).